Cages Driven Away from Equilibrium Binding by Electric Fields

Abstract

Molecules and materials that are pushed away from equilibrium can produce unique dynamic and adaptive properties that cannot be attained when they are at rest. In this issue of Chem, Cockroft and co-workers study the transient binding and debinding of coordination cages to the nanopocket of α-hemolysin and show that the binding and debinding events are driven out of equilibrium by an external electric field. Strong applied fields can invert the cage-nanopore binding selectivities and can enhance enantio-inversion to enrich one chiral form of the cage over the other. Molecules and materials that are pushed away from equilibrium can produce unique dynamic and adaptive properties that cannot be attained when they are at rest. In this issue of Chem, Cockroft and co-workers study the transient binding and debinding of coordination cages to the nanopocket of α-hemolysin and show that the binding and debinding events are driven out of equilibrium by an external electric field. Strong applied fields can invert the cage-nanopore binding selectivities and can enhance enantio-inversion to enrich one chiral form of the cage over the other. All living systems exist far from equilibrium, demanding a continuous influx of matter and/or energy for survival. For example, cytoskeletal microtubules, which can act as tracks for kinesin and dynein biomotors,1Alberts B. Johnson A. Lewis J. Raff M. Roberts K. Walter P. Molecular Biology of the Cell.Fifth Edition. Garland Science, 2008Google Scholar form by self-assembly of tubulin dimers but only when mediated by GTP (guanosine triphosphate).1Alberts B. Johnson A. Lewis J. Raff M. Roberts K. Walter P. Molecular Biology of the Cell.Fifth Edition. Garland Science, 2008Google Scholar Hydrolysis of GTP to GDP (guanosine diphosphate) and inorganic phosphate leads to the disassembly of microtubules, resulting in the collapse of its filament structure. Under physiological conditions, GTP-fueled polymerization and hydrolysis-driven depolymerization occur simultaneously such that their relative rates dictate net elongation or shrinking of the microtubules. This dynamic property arises when the system is not at equilibrium and is a ubiquitous feature of biology. Over the years, our growing understanding of natural and artificial systems sitting at thermodynamic equilibrium has allowed us to design new functional materials with high precision and predictability. However, the number of designed molecular systems displaying properties that emerge only when pushed away from equilibrium by the influx of energy and/or matter and that are inaccessible to equilibrium states is few and far between.2Rieß B. Boekhoven J. Applications of dissipative supramolecular materials with a tunable lifetime.ChemNanoMat. 2018; 4: 710-719Crossref Scopus (39) Google Scholar Some of the early examples have only been realized in recent years. For example, inspired by biological microtubules, van Esch, Eelkema, and co-workers reported the transient growth of one-dimensional supramolecular fibers3Boekhoven J. Hendriksen W.E. Koper G.J.M. Eelkema R. van Esch J.H. Transient assembly of active materials fueled by a chemical reaction.Science. 2015; 349: 1075-1079Crossref PubMed Scopus (461) Google Scholar upon consumption of chemical fuels and its sudden collapse upon removal of the fuel. In this issue of Chem, Cockroft and co-workers describe a far-from-equilibrium system4Borsley S. Haugland M.M. Oldknow S. Cooper J.A. Burke M.J. Scott A. Grantham W. Vallejo J. Brechin E.K. Lusby P.J. Cockroft S.L. Electrostatic forces in field-perturbed equilibria: nanopore analysis of cage complexes.Chem. 2019; 5: 1275-1292Scopus (12) Google Scholar involving one of the most elementary systems in supramolecular chemistry—that of the host-guest complex. One of the key features of this study is the ability to measure the time-dependent cage-nanopore binding events as blockages of the cis side of α-hemolysin5Song L. Hobaugh M.R. Shustak C. Cheley S. Bayley H. Gouaux J.E. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore.Science. 1996; 274: 1859-1866Crossref PubMed Scopus (1945) Google Scholar by using an external electric field. In so doing, the authors measure the on-off rates for binding as well as the equilibrium constants and thus use the same platform to drive the system away from equilibrium, verify the shift in populations, and identify new behaviors. The coordination cages used to block the cis opening of the nanopore are water-soluble M4L6 coordination cages (M, metal ion; L, ligand) bearing either positive or negative charges of different magnitudes (12−, 8−, 4−, 12+, 12+, 8+). The authors introduce the well-known α-hemolysin5Song L. Hobaugh M.R. Shustak C. Cheley S. Bayley H. Gouaux J.E. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore.Science. 1996; 274: 1859-1866Crossref PubMed Scopus (1945) Google Scholar as a nanopore into a lipid bilayer. Under an external transmembrane voltage applied across the bilayer, a characteristic current of ions flows through the nanopore of α-hemolysin. In this field-perturbed environment, the addition of tetrahedral cages on the cis side leads to blockage of the cis entrance at the single-molecule level. These binding events result in a temporary and discrete fluctuation of the transmembrane current. Following the ion current signatures of these binding events allows the authors to obtain the on-off rates (kon-koff) of cages with nanopores under external potential. As one might expect, positive and negative transmembrane potentials are used for measuring the binding of negatively and positively charged cages, respectively. Under an external electric field, the equilibrium constant, which is the ratio of the association and dissociation rates of cages with a nanopore (kon/koff = Ka), increases linearly with increased applied electric potential, as expected. The extrapolation of these data to zero applied potential provides the intrinsic value of the association constant. These extrapolated values suggest little to no binding of cages with the nanopore under non-perturbed conditions. The transient binding of cages with the nanopore also depends on the local electrostatic interactions between cages and the positively charged lysine residues at the cis entrance of α-hemolysin. As a result, the dissociation rate of negative cages from the nanopore (when positive applied potentials are used) is lower than that of positive cages (when negative applied potentials are used). Whereas the external electric field dictates the transient binding of cages, the local electrostatic interaction governs the dissociation of cages from the nanopores. This observation adds to the emerging interest in understanding how local electrostatics control elementary binding events.6Liu Y. Sengupta A. Raghavachari K. Flood A.H. Anion binding in solution: beyond the electrostatic regime.Chem. 2017; 3: 411-427Abstract Full Text Full Text PDF Scopus (100) Google Scholar, 7Hirsch B.E. McDonald K.P. Qiao B. Flood A.H. Tait S.L. Selective anion-induced crystal switching and binding in surface monolayers modulated by electric fields from scanning probes.ACS Nano. 2014; 8: 10858-10869Crossref PubMed Scopus (42) Google Scholar, 8Wang K. Cai X. Yao W. Tang D. Kataria R. Ashbaugh H.S. Byers L.D. Gibb B.C. Electrostatic control of macrocyclization reactions within nanospaces.J. Am. Chem. Soc. 2019; https://doi.org/10.1021/jacs.9b02287Crossref Scopus (45) Google Scholar The electric field can be used to turn on different behaviors. First, it is found to invert the selectivity of binding between the cages and the nanopore (Figures 1A and 1B). The competitive binding of the nanopore with 8− and 4− cages is tilted in favor of the 4− cages with a 150-fold excess. At low voltage (+50 mV), Le Chatelier’s principle is followed such that the binding of 4− cages is preferred over that of 8− cages. However, at higher potential (+150 mV), the binding selectivity is reversed, i.e., the nanopore binds the 8− cages preferentially. This effect arises from the higher charge density of the 8− cages, which debind less frequently than the 4− cage under the larger external field. This field drives the population of the bound states away from equilibrium. All the tetrahedral cages exist as a racemic mixture of two enantiomers, where all four metal centers are either ΔΔΔΔ or ΛΛΛΛ. In a previous publication,9Cooper J.A. Borsley S. Lusby P.J. Cockroft S.L. Discrimination of supramolecular chirality using a protein nanopore.Chem. Sci. 2017; 8: 5005-5009Crossref PubMed Google Scholar Lusby, Cockroft, and co-workers exploited the intrinsic chirality of the nanopore to discriminate the chirality of tetrahedral cages under an applied electric potential. Detection of the cages with different chirality benefits from the Ga3+-based cages’ retention of their chiralities at the applied voltage. In this issue of Chem,4Borsley S. Haugland M.M. Oldknow S. Cooper J.A. Burke M.J. Scott A. Grantham W. Vallejo J. Brechin E.K. Lusby P.J. Cockroft S.L. Electrostatic forces in field-perturbed equilibria: nanopore analysis of cage complexes.Chem. 2019; 5: 1275-1292Scopus (12) Google Scholar the authors observe an interesting electric-field-driven enantio-inversion of 8− tetrahedral cages based on Ge4+ metal centers (Figures 1C and 1D). This inversion occurs when the cages are bound inside the nanopore but only at an applied potential of +120 mV. At lower voltages, e.g., +80 mV, enantio-inversion events are very rare. The enantio-inversion of cages induced by the applied potential proceeds through meta-stable states, as seen in the transient current measurements. Interestingly, the ΔΔΔΔ cages exhibit more metastable events than the ΛΛΛΛ cages, leading to a net enhancement in ΛΛΛΛ cages. The single-molecule measurements, however, also verify the less-frequent enantio-inversion in the other direction from ΛΛΛΛ to ΔΔΔΔ. The chiral nanopocket of hemolysin most likely leads to this selectivity. In summary, this study illustrates the influence of an external electric field on the binding and debinding of charged cages with the nanopore of α-hemolysin. Given that the sizes and charges of the cages are synthetically modifiable, the authors understand and then control the transient behavior of the cages. The magnitude of the electric field is used to create out-of-equilibrium conditions to control the selective capture and release of cages and their stereo-inversion. In the future, it would be interesting to investigate whether it would be possible to exploit the interior of a cage, which has been well explored to capture a variety of guests,10Rizzuto F.J. von Krbek L.K.S. Nitschke J.R. Strategies for binding multiple guests in molecular coordination cages.Nat. Rev. Chem. 2019; 3: 204-222Crossref Scopus (207) Google Scholar to get unique functions under these non-equilibrium conditions: is it possible to selectively deliver a neutral guest residing inside a tetrahedral cage to the trans side of α-hemolysin? We envision that surprising new properties will continue to emerge when we learn how to control and study such “living” molecular systems. The authors acknowledge support from the National Science Foundation (CHE 1709909). Electrostatic Forces in Field-Perturbed Equilibria: Nanopore Analysis of Cage ComplexesBorsley et al.ChemApril 9, 2019In BriefSurprisingly little is known about the perturbing influence of externally applied fields on molecular-level processes that may drive a system away from equilibrium. Here, we investigated the effects of electric fields on molecular recognition events occurring at the single-molecule level. Using a nanopore-based approach, we showed that the applied electric field could also be used to drive processes including supramolecular enantio-inversion of metallosupramolecular cages or the capture and disassembly of cages within the nanopore. Full-Text PDF Open Archive